Effects of Cordyceps militaris fermentation products on reproductive development in juvenile male mice

Cordyceps militaris (CM) is a popular medicinal fungus; however, few studies have focused on its impact on the male reproductive system. We evaluated the effects of CM fermentation products on the reproductive development of juvenile male (JM) mice. Mice were divided into four experimental groups, each fed 5% CM products (weight per weight (w/w) in normal diet): extracellular polysaccharides (EPS), fermentation broth (FB), mycelia (MY), and whole fermentation products (FB plus MY, FBMY) for 28 days, while mice in the control group (CT) were fed a normal diet. Basic body parameters, testicular structure, sperm parameters, and sex hormones concentrations were analyzed. Compared to the CT group, mice in the EPS, MY, and FBMY groups showed a significantly increased mean seminiferous tubule area (p < 0.05), mice in the FB and MY groups had significantly higher sperm concentrations (p < 0.05), and mice in the EPS, FB, and FBMY groups showed significantly increased ratios of motile sperm (p < 0.05). Meanwhile, EPS significantly promoted the ability of JM mice to synthesize testosterone (p < 0.05). Furthermore, all CM products significantly increased the food intake of JM mice (p < 0.05) but did not significantly change their water intake and body weight gain (p > 0.05). In conclusion, CM products, especially EPS, exhibit strong androgen-like activities that can promote male reproductive development.

Effects of C. militaris fermentation products on food and water intake and body weight gain. Food and water intake and body weight gain in mice of all groups are presented in Fig. 1. The food intake of JM mice in the experimental groups fed with C. militaris fermentation products (EPS, FB, MY, and FBMY) was significantly higher than that of the CT group that were fed a normal diet (all p < 0.05; Fig. 1A). JM mice in the EPS group had the highest food intake (p < 0.001 vs. CT, p = 0.007 vs. FB, p = 0.034 vs. MY, p = 0.343 vs. FBMY). A possible explanation for this phenomenon is that the sweetness of EPS increases the palatability of the pellets. Moreover, there were no significant differences in water intake (Fig. 1B) or body weight gain for 28 days (Fig. 1C) between the experimental groups and the CT group (all p > 0.05). Although mice in the EPS group ate more than mice in the other groups, their body weight gains were only significantly increased when compared with the FB group (p < 0.05). These results imply that body weight gain in JM mice is controlled or regulated by EPS addition, thereby preventing obesity.
Recently, the regulatory role of some medicinal fungi on obesity has been gradually clarified through 16S rRNA gene sequencing and metabolomic studies [17][18][19][20][21][22] . Since intestinal bacteria can not only increase the absorption of intestinal monosaccharides and short-chain fatty acids and promote the fat accumulation in the liver tissue 23 , but the lipopolysaccharide of gram-negative bacteria can also bind to receptors on the surface of immune cells, causing an inflammatory response thereby aggravating obesity 24 . Previous studies have demonstrated that Table 1. Main components of the four C. militaris fermentation products used in this study. *N.D. means the detection result is below limit of quantitation, i.e., not detected. Panels marked with different lowercase letters are significantly different (p < 0.05).  25 . C. militaris polysaccharides could increase gut bacteria negatively associated with obesity traits, decrease the population of gram-negative bacteria, effectively improve intestinal flora dysbiosis in HFD-induced mice, and regulate the levels of metabolites, thereby reducing body weight, fat accumulation, and pro-inflammatory cytokine levels in mice 26,27 . Selenium-enriched C. militaris could reduce serum triglyceride and low-density lipoprotein cholesterol levels, inhibit serum lipopolysaccharide-binding proteins, adiponectin levels, and pro-inflammatory gene expression in HFD mice, and improve the expression of related anti-inflammatory genes 28 . Although the mechanisms remain to be elucidated, C. militaris fermentation products, especially EPS, may effectively prevent obesity during the development of JM mice by modulating the intestinal flora.

Effects of C. militaris fermentation products on apparent testicular parameters. Testicular vol-
umes and weights of JM mice in each group are shown in Fig. 2. There were no significant differences in testicular volume between the experimental and CT groups (all p > 0.05; Fig It has been reported that cordycepin reduces fat accumulation around the epididymis of HFD rats 29 and significantly reduces epididymal adipocyte sizes in a dose-dependent manner 30 . In addition, C. militaris polysaccharides can decrease epididymal fat index by modulating the expression of key genes and proteins in epididymal fat 31 , including reducing sterol regulatory element binding protein-1c level by approximately 49%, inhibiting the expression of peroxisome proliferator-activated receptor γ protein by approximately 67% 32 , thereby significantly reducing epididymal adipogenesis. This suggests that the decreased testicular weight of JM mice in the EPS group may be related to the fact that EPS can reduce lipid and adipocyte differentiation in testis and surrounding tissue. Since the testes play crucial roles in producing sperms and synthesizing androgens, the testicular structure, sperm parameters, and sex hormone levels in sera were further evaluated. Effects of C. militaris fermentation products on testicular morphology and structure. The hematoxylin and eosin (H&E) staining images of testicular sections of JM mice from each group are shown in Fig. 3. The morphology and structure of the seminiferous tubule (STs) were normal, and germ cells in the STs were in a regular arrangement in all groups. In the same field of view, STs were larger, and sperm cell densities were higher in all experimental groups than in the CT group. These results indicate that C. militaris fermentation products promote ST development in the testes of JM mice. Therefore, we evaluated the effect of C. militaris fermentation products on the morphological indicators of STs.
The number and mean area of the STs of JM mice in each group are presented in Fig. 4. The number of STs in all experimental groups was significantly lower than that in the CT group (p < 0.05; Fig. 4A). Mice in the EPS group had the lowest number of STs (p < 0.001 vs. CT, p < 0.001 vs. FB, p = 0.761 vs. MY, p = 0.052 vs. FBMY). Moreover, the mean area of STs in all experimental groups, except for the FB group, was significantly larger than that in the CT group (all p < 0.05; Fig. 4B). The mean area of STs was the largest in the MY group (p < 0.001 vs. CT, p = 0.221 vs. EPS, p < 0.001 vs. FB, p = 0.025 vs. FBMY). The larger the mean area of the STs, the more mature the STs.
The STs of the testis are the site of spermatogenesis and are composed of spermatogenic cells and Sertoli cells, the former producing sperm and the latter supporting and nourishing spermatogenic cells 33 . In this study, except that EPS significantly reduced testicular weight, C. militaris fermentation products did not significantly affect testicular volumes or weights of JM mice, but significantly increased the size and maturation of STs. These results suggest that C. militaris enhances testicular development, thereby promoting spermatogenesis in JM mice.  Table 2). Sperm concentrations in JM mice in all experimental groups were higher than in the CT group. Mice in both the FB and MY groups showed significant increases in sperm concentrations compared to the CT group (p < 0.05). Mice in the MY group had the highest sperm concentrations. Furthermore, the ratio of motile sperm was also higher in all experimental groups than in the CT group. Except for the MY group, the motile sperm ratios in all experimental groups were significantly higher than in the CT group (all p < 0.05). Ratios of motile sperm were the highest in the FBMY group.
Based on the results shown in Figs. 3, 4 and Table 2, C. militaris fermentation products promoted the development and maturation of STs, enhanced spermatogenesis in the testes, and increased mature sperm concentration and motile sperm ratio in the epididymis. These results are consistent with reports showing increased sperm concentrations and motilities in boars 34 and Sprague-Dawley rats 35 following MY supplementation.
Oxidative stress is an important cause of testicular damage, which can lead to testicular dysplasia, spermatogenic cell necrosis and apoptosis, resulting in male reproductive dysfunction 36 . C. militaris is a natural antioxidant with excellent ability to scavenge reactive oxygen radicals 37 . Among them, cordycepin can regulate the activities    39 , effectively improving sexual dysfunction in aged rats. Moreover, another study showed that the EPS extracted from the C. militaris FB could promote the proliferation of spleen T and B lymphocytes through MAPK signaling 40 ; thus, EPS may also be able to resist testicular oxidation through this pathway. Therefore, the antioxidant activities of C. militaris may explain why these fermented products can promote male reproductive development in JM mice.

Effects of C. militaris fermentation products on serum sex hormone levels.
To investigate the effects of supplementation with C. militaris fermentation products on male reproductive development, the serum sex hormone levels of JM mice were analyzed (Fig. 5). Although mice in both the EPS and FB groups showed higher testosterone (T) concentrations than those in the CT group, there were no significant differences in T concentrations between each experimental group and the CT group (all p > 0.05; Fig. 5A). Mice in the EPS group had the highest T levels. However, T levels in mice in the FBMY group were significantly lower than those in the other experimental groups (all p < 0.05), but not in the CT group. Since estradiol (E2) levels may antagonize the effects of T, serum E2 levels were also analyzed. Although mice in both the FB and MY groups showed higher E2 concentrations in sera than those in the CT group, there were no significant differences in E2 concentrations between each experimental group and the CT group (all p > 0.05; Fig. 5B). However, E2 levels were the lowest in the FBMY group but only significantly different when Table 2. Effects of C. militaris fermentation products on sperm parameters in epididymis of JM mice. *Values are presented as means ± SD (n = 5 for all test groups). Panels marked with different lowercase letters are significantly different (p < 0.05).  www.nature.com/scientificreports/ compared to the FB and MY groups (both p < 0.05). In males, E2 is mostly converted through the aromatization of T 41 . The results shown in Figs. 5A,B support the positive correlations between T and E2 levels, and the lower E2 concentration, resulting in lower testicular volumes, number of STs, and sperm concentrations in epididymis of mice in the FBMY group. However, the reason for the lower T and E2 levels in the FBMY group than in the other groups remains to be elucidated. Since luteinizing hormone (LH) is secreted by the anterior pituitary gland, which stimulates ovulation in females and androgen synthesis in males, serum LH levels were analyzed. Except for the FB group, there were no significant differences in LH concentrations among the other groups (all p > 0.05; Fig. 5C). However, the LH levels were significantly lower in the FB group than in the CT, EPS, and MY groups (all p < 0.05). The synthesis and secretion of sex hormones are regulated mainly by the hypothalamus-pituitary-gonadal axis. Thus, increases in both T and E2 levels in the FB group may have a negative effect on the LH levels secreted by the pituitary gland. This explains why mice in the FB group had the lowest LH levels.
Since T concentrations in testes are at least 25-100 times higher than those in sera 42 , the ratios of serum T levels to testicular weights (T/T) were calculated to directly assess the ability of testes to synthesize T. Results showed that T/T ratios were higher in all experimental groups, except the FBMY group, than those in the CT group (Fig. 5D). The T/T ratios were the highest in the EPS group and showed significant differences when compared with the CT, MY, and FBMY groups (all p < 0.05). Moreover, the T/T ratios of the FBMY group were the lowest and showed a significant decreases when compared with those of the EPS and FB groups ( p < 0.05), but there was no significant difference with the CT group.
The results shown in Fig. 5 indicated that C. militaris fermentation products, especially EPS, promote the development of testes and elevate serum levels of T and T/T ratios in JM mice. Our findings agree with one previous study that demonstrated a C. militaris-induced increase in T productions in Sprague-Dawley rats 43 . Another study showed that O. sinensis stimulates T productions in vivo and in vitro 44 . Polysaccharides and glycoproteins of O. sinensis share structural similarities with LH and can be recognized by LH receptors on Leydig cells to stimulate T productions. Although the specific mechanism remains unknown, C. militaris fermentation products, especially EPS, showed androgen-like activity, which can promote male reproductive development in JM mice.
The results shown in Figs. 2, 3, 4 and 5 and Table 2 indicate that C. militaris fermentation products, especially EPS, promote the development of testes, elevate serum levels of T and T/T ratios, and then increase sperm concentrations and ratios of motile sperms in the epididymis of JM mice. Since all mice in this study were fed ad libitum, it is possible that the EPS-containing feed increased the mice's appetite due to its sweetness. From our results, increased EPS intake may better promote reproductive development in male JM mice. In addition, C. militaris extract has been reported to prevent obesity and fat accumulation in ovariectomized rats, lower triglyceride levels, and enhance estrogen receptor agonistic activity and phosphorylation, effectively regulating menopause-induced obesity 45 . Therefore, these findings suggest that C. militaris fermentation products can promote the reproductive system health and prevent metabolic syndromes including obesity.

Conclusion
In this study, we evaluated the effects of four different C. militaris fermentation products on male reproductive development in JM mice. EPS showed the strongest androgen-like activity, which promoted male reproductive development. EPS supplementation can increase the mean area and maturation of STs in the testes, sperm concentrations and ratios of motile sperms in the epididymis, and serum T levels and T/T ratios. Therefore, EPS supplementation can effectively improve male reproductive development in JM mice, and may prevent or assist in treating male reproductive dysfunction or infertility. Furthermore, it is important to note that C. militaris fermentation products can effectively control or regulate body weight gain in JM mice during the experimental period of 28 days, thus it has the potential to assist in obesity treatment. However, it must be noted that clinical studies are required to determine if these beneficial effects of C. militaris can apply to humans.

Materials and methods
Materials. C. militaris fermentation products including EPS, FB, and MY, were gifts from Dalong Biotechnology Co. Ltd. (Taichung, Taiwan). EPS were obtained from ethanol precipitation of the FB, centrifugation, and freeze-drying. The yields of polysaccharides, cordycepin, adenosine, and proteins of the EPS, FB, MY and whole fermentation product (FBMY) were detected by the phenol-sulfuric acid method 46 high-performance liquid chromatography (HPLC) method 47 , and Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions, respectively ( Table 1). The feed pellets of different experimental groups were produced by mixing 5% (w/w) dry weight of EPS, FB, MY, and FBMY with 95% Laboratory Rodent Diet 5001.
Animals. Three-week-old male mice (C57BL/6JNarl), 25 in number, were purchased from the National Laboratory Animal Center (Taipei, Taiwan). During the experimental period, they were kept in an air-conditioned room at 24 ± 2 °C and 50-60% humidity with a 12 h light/dark cycle. They were randomly divided into five groups (n = 5 each). Mice in the control (CT) group were fed Laboratory Rodent Diet 5001 only, while mice in the experimental groups were fed pellets containing 5% (w/w) EPS, FB, MY, and FBMY for 28 days. Mice were sacrificed using the cervical dislocation method after anesthesia with isoflurane (Baxter, CA, USA) at the end of the experiment. Blood samples were obtained by cardiac puncture. In addition, testes and epididymides were removed for measurement and analysis. H&E staining of testis sections. Each right testis was fixed in a 10% neutral formalin solution (Leica Surgipath Inc., Buffalo Grove, IL, USA) for more than 16 h. After fixation, testes were cut in half at the widest point and embedded in paraffin. Five-micrometer histological sections were deparaffinized, rehydrated, and stained